Project Summary This proposal describes a plan to develop a method for visualizing live cell physiology with high resolution using integrated Differential Phase Contrast-Scanning Transmission Electron Microscopy (iDPC-STEM) at low dose to promote viability. Visualizing physiology demands spatial resolution with a commensurate depth-of- field on the scale of the protein machinery (3-7 nm) that drives it without concomitant damage. With the introduction of a liquid flow cell containing water in a vacuum-tight envelope made from membranes that are transparent to the electron beam, it should be possible to scrutinize biology with high-resolution under physiological conditions with STEM. This proposal focuses on three specific technical challenges, testing solutions in a crucible of well characterized biological systems: 1. Improve resolution using a liquid flow cell formed from ultra-thin membranes and thin spacers. To reduce scattering in the membrane and liquid, it is practical to shrink the silicon nitride (SiN) membranes forming the liquid cell to 8-10 nm, and space them 150 nm apart without compromising the window integrity. To eliminate bulging in a liquid cell loaded with fluid, the windows will be reinforced with thick ribs so that a large >400 ?m2 area can be spanned. However, even 10 nm SiN membranes are still too thick for high-resolution imaging. So, (3 nm) thin amorphous silicon (a-Si) and atomically thin graphene or h-BN membranes spanning ribs formed from SiN will be used as windows for high-resolution imaging. The resolution will be tested using a Titan STEM by visualizing adenosine triphosphate (ATP) and fluorescent streptavidin (STR). 2. Improve contrast with iDPC-STEM imaging. To increase the visibility of transparent biological samples, a phase-contrast method for imaging, iDPC-STEM, will be adopted that uses a four-quadrant (segmented) split- detector to measure the gradient of a phase object. iDPC-STEM boasts a higher signal to noise ratio compared to conventional STEM, which offers the possibility for extremely low-dose imaging. The resolution, contrast and concomitant damage will be tested in an aberration-corrected, iDPC-equipped Themis Z (with 60 pm resolution) by visualizing ATP and fluorescent STR in thin (0-50 nm thick) liquid layers. 3. Finally, low-dose iDPC-STEM will be used with an ultra-thin liquid flow cell to visualize the smallest prokaryotic cells. If the electron probe interacts with a cell at the top membrane in the liquid cell, high- resolution images may be captured this way. Because the probe is so shallow along the optic-axis, a focus series may also be used to section a cell for 3D tomography. To test these ideas, four strains of Mycoplasma (100 nm in size) will be cultured in a shallow (150 nm) flow cell and visualized with iDPC-STEM to discover the role their nanostructure plays in infection. In specimens this thick, multi-slice simulations may be required to inform on the structure. After exposure to the beam, a LIVE/DEAD assay, along with Mycoplasma transformed with plasmids that produce an inducible fluorescent reporter will be used to score viability.